Biomolecule and/or cellular arrays on metal surfaces

ABSTRACT

Disclosed is a process to construct multi-component biomolecule or cellular arrays suitable for use in SPR imaging studies of large molecule, cellular/molecular, and cell/cell interactions. Also disclosed are the resulting arrays. The success of the procedure hinges on the use of a reversible protecting group to modify reversibly ω-functionalized alkanethiols self-assembled on metal substrates. The arrays themselves include a metal substrate, a continuous layer of an identical ω-modified alkanthiol adhered to the metal substrate, and one or more discrete spots of biomolecules or cells directly bonded to the continuous layer of ω-modified alkenthiol. The areas of the continuous layer of ω-modified alkenthiol not covered by one of the discrete spots are covered by a background material resistant to non-specific protein binding.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 09/456,038, filed Dec. 3,1999 (now U.S. Pat. No. 6,489,102, issued Dec. 3, 2002), which is adivisional of application Ser. No. 09/368,991, filed Aug. 5, 1999 (nowU.S. Pat. No. 6,127,129, issued Oct. 3, 2000), both of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention is directed to the fabrication of biomolecular or cellulararrays on metal surfaces for use in the study of interactions betweenlarge molecules, between cells and large molecules, and between cells,such as nucleic acid-protein interactions or cellular interactions withantigens.

DESCRIPTION OF THE PRIOR ART

The binding of proteins to DNA plays a pivotal role in the regulationand control of gene expression, replication and recombination. Inaddition, enzymes that recognize and modify specific oligonucleotidesequences are critical components of biological nucleic acidmanipulation and repair systems. An enhanced understanding of how theseproteins recognize certain oligonucleotide sequences would aid in thedesign of biomedical systems which could, for example, be used toregulate the expression of therapeutic proteins. For this reason, thestudy of protein-nucleic acid interactions (i.e., protein-DNA andprotein-RNA interactions) is a rapidly growing area of molecularbiology, aided in part by recent advances in NMR and X-ray structuraldetermination methods. At the same time, the explosive increase in theamount of available genomic and extra-genomic (i.e., ribosomal) sequenceinformation obtained from large-scale nucleic acid sequencing effortscreates a need to survey this vast amount of new sequence data forprotein binding sites. The present invention addresses this need byusing surface plasmon resonance (SPR) imaging techniques as a rapid andefficient method for screening the sequence or structure-specificbinding of proteins to large arrays of nucleic acid moleculesimmobilized at chemically-modified metal surfaces.

Arrays of DNA molecules attached to planar surfaces are currentlyemployed in hybridization adsorption experiments to sequence DNA, Peaseet al. (1994) Proc. Natl. Acd. Sci. USA 91:5022–5026; to screen forgenetic mutations, Winzeler et al. (1998) Science 281:1194–1197: and inDNA computing applications, Frutos et al. (1997) Nucleic Acids Res.25:4748–4757; and Frutos et al (1998) J. Am. Chem. Soc. 120:10277–10282.These arrays are exposed to solutions containing fluorescently labeledcomplementary DNA sequences, rinsed, and then “read-out” usingfluorescence imaging methods.

The technique of surface plasmon resonance (SPR) is a surface-sensitive,optical detection method well suited to the monitoring of reversible,protein-nucleic acid interactions. The commercially successful “BIAcore”SPR instrument (Biacore AB, Uppsala, Sweden) has been used previously,for example, to study the interaction of DNA molecules with variousenzymes. Although powerful, the “BIAcore” instrument has no imagingcapabilities. This severely limits the number of DNA sequences that canbe screened in a single experiment.

Surface plasmon resonance (SPR) is a surface optical technique which issensitive to the thickness and index of refraction of material at theinterface between a free electron metal (e.g. gold, silver, copper,cadmium, aluminum) and a bulk medium, such as air or water. Surfaceplasmon resonance may be achieved by using the evanescent wave which isgenerated when a laser beam linearly polarized parallel to the plane ofincidence impinges onto a prism coated with a thin metal film. The metalmay also be coated onto a thin transparent substrate such as glass, andthis glass brought into optical contact with the prism. SPR is mosteasily observed as a reduction of the total internally reflected lightjust past the critical angle of the prism. This angle of minimumreflectivity (denoted as the SPR angle) shifts to higher angles asmaterial is adsorbed onto the metal layer. The shift in the angle can beconverted to a measure of the thickness of the adsorbed or addedmaterial by using complex Fresnel calculations and can be used to detectthe presence or absence of materials on top of the metal layer.

In using SPR to test for biological, biochemical, or chemicalsubstances, a beam of light from a laser source is directed through aprism onto a biosensor consisting of a transparent substrate, usuallyglass, which has one external surface covered with a thin film of anoble metal, which in turn is covered with an organic film thatinteracts strongly with an analyte, such as a biological, biochemical,or chemical substance. The organic film can contain substances, such asantibodies or antigens, which can bind with an analyte in a sample tocause an increased thickness which will shift the SPR angle. Bymonitoring either the position of the SPR angle or the reflectivity at afixed angle near the SPR angle, the presence or absence of an analyte inthe sample can be detected.

Various types of equipment for using SPR with a biosensor for biologicalor biochemical or chemical substances are described by the Liedberg etal. article found in “Sensors and Actuators,” Vol. 4, 1983, page 299.See also European Patent Application 0 305 108 and U.S. Pat. No.5,374,563.

The use of conventional SPR as a testing tool offers several advantagesand disadvantages. For example, it is relatively fast, it requires nolabeling, and it can be performed on site. However, as noted above,commercially-available devices, such as the “BIAcore” instrument, offerno imaging capabilities. Additionally, to achieve the high through-putdemanded by large-scale users, there is a need for a simple, practicalbiosensor which can be readily modified or adapted to test a widevariety of compounds simultaneously.

In SPR imaging, a light source (typically a HeNe laser) is used toilluminate a prism/thin gold film sample assembly at an incident anglethat is near the SPR angle, and the reflected light is detected at afixed angle with a CCD camera to produce an SPR image. The SPR imagearises from variations in the reflected light intensity from differentparts of the sample; these variations are created by any changes inorganic film thickness or index of refraction that occur upon adsorptiononto the modified gold surface. Since SPR imaging is sensitive only tomolecules in close proximity to the surface (within ˜200 nm), unboundmolecules remaining in solution do not interfere with in situmeasurements.

The formation of robust, reproducible arrays of oligonucleotidestethered to metal-coated surfaces (most often gold) is an essentialrequirement for SPR imaging of protein-nucleic acid bindinginteractions. To use SPR imaging techniques, it is essential that thenucleic acid array be constructed on a noble metal surface, and for thisreason DNA arrays on glass supports from commercially available sourcessuch as Affymetrix (Santa Clara, Calif.) are not a viable option. Usingself-assembled monolayers of substituted alkanethiols as a startingpoint, others have previously developed schemes to attachsingle-stranded DNA molecules to chemically modified gold surfaces. See,for instance, U.S. Pat. No. 5, 629,213). In the subject invention,however, UV photopatterning and microcontact printing techniques arebrought to bear to allow alkanethiols to be assembled in a site-directedmanner on the metal surface, thereby enabling the creation ofmulti-component arrays. A combination of these processing techniquesalong with novel surface chemical reactions enables the manufacture ofnucleic acid arrays as described herein.

SUMMARY OF THE INVENTION

Disclosed is a multi-step chemical modification procedure to createbiomolecule and/or cellular arrays on metal substrates, the arrays beingspecifically tailored for the study of biomolecular and cellularinteractions using surface plasmon resonance imaging. Arrays fabricatedby this procedure meet three specific requirements, namely (i) thebiomolecules are covalently attached to the surface and remain activeand accessible to hybridization and protein binding; (ii) the arraybackground is, at first, sufficiently hydrophobic so as to allow for the“pinning” of aqueous solutions of biomolecules or cells at specificarray locations; and (iii) the final array background acts to inhibitthe non-specific binding of protein molecules to the surface. The keycomponents of this fabrication scheme are the utilization of areversible hydrophobic protecting group, preferably Fmoc, to control thesurface hydrophobicity of a tethered ω-modified alkanethiol monolayerand the attachment of a poly(ethylene glycol) (PEG) group to render thesurface protein resistant. Polarization-modulation Fourier Transforminfrared (PM-FTIR) spectroscopy, contact angle, and SPR measurements areused to characterize each step in the surface modification procedure andconfirm that the array background inhibits the nonspecific binding ofproteins. As a final test, an SPR imaging experiment which measures theadsorption of single-stranded DNA binding protein (SSB) to a dualcomponent, oligonucleotide array demonstrates the utility of thesesurfaces for the monitoring of protein-nucleic acid interactions.

The multi-step procedure disclosed herein is used to create an array ofspots that are surrounded first by a hydrophobic background which allowsfor the pinning of aqueous biomolecule or cell solutions onto individualarray elements and then to replace the hydrophobic background with onethat resists the non-specific adsorption of proteins during in situ SPRimaging measurements, thereby yielding an array of biomolecule or cell“islands” in a “sea” which resists non-specific adsorption of proteins.

In the preferred embodiment, amine-terminated alkanethiol monolayers areemployed as the base layer, and Fmoc and PEG modifiers are used tocreate the sequentially hydrophobic and protein adsorption-resistantsurfaces, respectively. In the preferred embodiment, the chemicalmodification steps are: (i) the adsorption and self-assembly of an11-mercaptoundecylamine (MUAM) monolayer on an evaporated gold thinfilm; (ii) the reaction of the MUAM monolayer with an Fmoc protectinggroup to create a hydrophobic surface; (iii) the photopatterned removalof the alkanethiol followed by (iv) the re-adsorption of MUAM to createan array of MUAM squares (approximately 750 μm×750 μm, although smalleror larger squares are attainable) surrounded by a hydrophobic MUAM-Fmocbackground that can pin drops of aqueous solution; (v) the attachment ofoligonucleotide sequences onto the MUAM squares by the reaction of theamine-terminated surface with the heterobifunctional cross-linker(preferably SSMCC), followed by a coupling reaction to a small volume(0.1 μL) of thiol-modified DNA; (vi) the removal of the Fmoc protectinggroup followed by (vii) a pegylation reaction of the MUAM with PEG-NHSto create a protein adsorption-resistant background.

A combination of polarization-modulation FTIR spectroscopy, contactangle and scanning angle SPR measurements are used to characterize thesurface modification procedure. An SPR imaging measurement—of theadsorption of single-stranded DNA binding protein (SSB) onto anoligonucleotide array created by this procedure is used to demonstratethe utility of these surfaces to probe nucleic acid interactions withprotein and other analytes.

A primary advantage of the subject invention is that it allows an arrayof immobilized biomolecules or cells to be constructed in which each“island” of bound molecules or cells may differ from the other islandsin the array. This allows for massive and simultaneous analysis of atremendous number of different molecules or cells for their individualaffinities and/or binding characteristics to a selected analyte. Thefabrication method described herein is well-suited to automation and SPRexperiemnts can be analyzed using standard-format microtiter plates andlab automation equipment (ie., 96-well, 384-well, and larger formats).

The arrays described herein are useful for any number of analyseswherein a biomolecule or cell interacts with a protein, antigen, or someother molecule, such as in determining binding affinities, epitopemapping, restriction site mapping, measuring the binding effects ofshort-range secondary structure in nucleic acids, etc. For example, bybuilding an array wherein islands of nucleic acids differsystematically, as by length or primary sequence, the interactions ofany given nucleic acid sequence for any given analyte can be quickly andexhaustively investigated. Likewise, the effects of short-rangesecondary structure in nucleic acids can be investigated by building anarray wherein the islands of nucleic acids differ in sequence such thatthe islands contain nucleic acid sequences which progressively containmore stable secondary structures and then scanning the array afterexposure to a given analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a fabrication scheme forconstructing multi-element DNA arrays according to the presentinvention. A clean gold surface is reacted with an amine-terminatedalkanethiol and subsequently reacted with a protecting group to create ahydrophobic surface. This surface is then exposed to UV radiationthrough a quartz mask and rinsed with solvent to remove thealkanethiol+protecting group from specific areas of the surface, leavingbare gold pads. These bare gold areas on the sample surface are filledin with the alkanethiol, resulting in an array of alkanethiol padssurrounded by a hydrophobic protecting-group background. Solutions ofnucelic acid are then delivered by pipette onto the specific arraylocations and are covalently bound to the surface via a bifunctionallinker. In the final two steps, the protecting groups on the arraybackground are removed and replaced by functional groups which prohibitthe non-specific binding of analyte proteins to the background.

FIG. 2 depicts a surface reaction scheme showing the steps involved inthe reversible modification of the array background. The startingamine-terminated alkanethiol surface is reacted with the Fmoc-NHSprotecting group to form a carbamate linkage thus creating a hydrophobicFmoc-terminated surface. After nucleic acid immobilization, the surfaceis deprotected, resulting in the return of the original alkanethiolsurface. In the final array fabrication step, the deprotectedalkanethiol surface is reacted with PEG-NHS to form an amide bond whichcovalently attaches PEG to the array surface (to prevent non-specificbinding of analyte proteins to the background).

FIG. 3 depicts PM-FTIRRAS spectra in the mid-IR region for the surfacesinvolved in the array background modification. (A) The starting MUAMsurface. (B) After reaction with Fmoc-NHS, bands indicative of thecarbamate linkage and the Fmoc ring stretch appear in the spectrum. (C)The surface is deprotected and reverts back to the MUAM surface asevidenced by the similarities between spectra A and C. (D) Afterreaction with PEG-NHS, bands indicative of the amide linkage as well asthose associated with the ethylene glycol groups are present.

FIG. 4 depicts a surface reaction scheme showing the steps involved inimmobilizing biomolecules, in this case DNA, to the array locations. Abifunctional linker, such as SSMCC, is used to link thiol-modified DNAto the MUAM pads.

FIG. 5 depicts a series of line profiles showing in situ hybridizationand the adsorption of single stranded DNA binding protein (SSB) onto adual component DNA array containing oligonucleotide sequences D1 and D2.The solid line is the percent reflectivity measured for the startingsurface composed of alternating DNA probe spots D1 and D2. The dashedline is the % R measured after exposing the surface to a solutioncontaining the complement to D2. Apparent is an increase in % R atposition D2 upon binding of the complementary DNA sequence. Thedot-dashed line is the % R measured after exposing the surface to a 200nM solution of SSB. While measurable binding did occur at array locationD2 (which contained double stranded DNA), the protein clearly bound moreabundantly to the single-stranded sequence D1.

FIG. 6 depicts an in situ SPR difference image showing the binding ofsingle stranded DNA binding protein (SSB) to a checkerboard array ofsingle- and double-stranded oligonucleotide sequences. Images collectedimmediately before and after exposure of the surface to SSB weresubtracted to produce the image shown. Significant binding of theprotein to array locations with covalently bound single-stranded DNAsequences occurred, whereas very little binding occurred at the arraylocations which contained double-stranded DNA sequences.

FIG. 7 depicts a series of line profiles showing the in situ adsorptionof bovine serum albumin (BSA) onto a patterned C₁₈/MUAM+PEG surface. Thesolid line is the % R measured for a surface array of 350 mm spots ofC₁₈ surrounded by a pegylated MUAM background. The dotted line is the %R measured after exposing the surface to a 1 mg/mL solution of BSA. Themuch lower change in % R for the MUAM-PEG regions indicates that thepegylated background is much more efficient than C₁₈ in resisting thenon-specific binding of the BSA.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Commercial Suppliers:

The following abbreviations and terms are used throughout thespecification and claims. All other terms have their standard, acceptedmeaning in the relevant art.

“biomolecule”=any molecule found in biological material, expresslyincluding, but not limited to nucleic acids, proteins, peptides,antibodies, enzymes, cell-wall components such as phospholipids, etc.,and modified and synthetic forms thereof, such as labeled biomoleculesand recombinant biomolecules.

“BSA”=bovine serum albumin (Sigma Chemical, St. Louis, Mo.).

“DMF”=dimethylformamide.

“Fmoc-NHS”=9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Novabiochem,La Jolla, Calif.).

“metal substrate” or “metal film”=a noble-metal thin film (gold, silver,copper, platinum, etc.). Gold is preferred.

“MUAM”=11-mercaptoundecylamine (a generous gift from the laboratory ofProfessor George M. Whitesides, Harvard University, Boston, Mass.).

“NHSS”=N-hydroxysulfosuccinimide ester.

“nucleic acids”=deoxyribonucleic acids (DNA), ribonucleic acids (RNA),and peptide nucleic acids from any source, and modified forms thereof,including, without limitation, labeled (radioactive, fluorescent, etc.)nucleic acids, and nucleic acids modified to include a binding moietysuch as a thiol group or a biotin tag.

“PEG”=poly(ethylene glycol).

“PEG-NHS”=N-hydroxysuccinimidyl ester of methoxypoly(ethylene glycol)propionic acid MW 2000 (Shearwater Polymers, Inc., Huntsville, Ala.).

“poly(ethylene glycol)-modified alkanethiol”=HS(CH₂)₁₁(OCH₂CH₂)₃OH (fromDr. Whitesides' laboratory).

“SSB”=single-stranded DNA binding protein (Pharmacia Biotech,Piscataway, N.J.).

“SSMCC”=sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Pierce Chemical, Rockford, Ill.).

“TAEA”=tris(2-aminoethyl)amine (Aldrich Chemical, Milwaukee, Wis.).

“TEA”=triethanolamine hydrochloride (Sigma)

“ω-modified alkanethiol”=an alkanethiol whose terminal carbon atom hasbeen modified by the addition of a chemically-reactive moiety such as anamino, hydroxy, carboxy, or thiol moiety.

The above chemicals and were all used as received. Solvents were ofstandard laboratory grade and Millipore (Marlborough, Mass.) filteredwater was used for all aqueous solutions and rinsing.

The chemical modification of a metal substrate to create a nucleic acidarray thereon proceeds in seven general steps These steps areillustrated schematically in FIG. 1 and are as follows:

(1). Self-assembly of an ω-modified alkanethiol monolayer on a metalsubstrate. The ω-modification to the alkanethiol may be the addition ofany moiety with enables further covalent linkages to be made theω-terminus of the alkanethiol. Such modifications include, withoutlimitation, the addition of an amine group, a hydroxyl group, a carboxylgroup, or a thiol group to the ω carbon of the alkanethiol chain. Thealkanethiol monolayer is preferably an amino-C₈–C₂₄-alkanethiol, astraight-chain alkane being much preferred to branched alkane; the mostpreferred ω-modified alkanethiol is MUAM.(2). Reaction of the ω-modified alkanethiol surface with a hydrophobicprotecting group, most preferably Fmoc.(3). Photopatterning of the surface to create an array of bare metalareas.(4). Re-assembly using additional ω-modified alkanethiol to fill in thebare metal array elements, thereby yielding islands of ω-modifiedalkanethiol.(5). Covalently attaching biomolecules or cells to the islands ofω-modified alkanethiol.(6). Removal of the protecting group from the array background.(7). Reaction of the background with a material, preferably PEG, to makethe background resistant to non-specific protein binding.(The numbers in parentheses directly above are reference numerals inFIG. 1.)

To ensure the quality of the finished product, each of the above stepsmay be monitored using PM-FTIRRAS, contact angle measurements, andscanning-angle SPR.

The above steps are now described in greater detail, with specificreference being made to FIG. 1.

Step (1). In step (1), a monolayer of ω-modified alkanethiol, preferablyan amine-terminated alkanethiol, most preferably MUAM, is self-assembledfrom an ethanolic solution onto a silanized substrate (glass or othersubstrate transparent to the wavelengths of radiation to be used insubsequent analysis) coated with a thin noble-metal film. In thepreferred embodiment, a film of gold about 450 Å thick is used. Thethickness of the metal film is not overly critical insofar as the filmis uniformly applied and will function in SPR imaging analysis.Self-assembled monolayers of ω-modified alkanethiols on gold have beendescribed previously, see, for example, Thomas et al. (1995) J. Am.Chem. Soc. 117:3830–3834, and are generally accepted by most to formwell-ordered, monomolecular films. However, if left exposed for extendedperiods of time, the terminal amine groups of amino-modified alkanthiolswill react with CO₂ to form carbamate salts on the surface.Consequently, amino-terminated alkanethiol-coated substrates should behandled with care and their exposure to CO₂ minimized.

The PM-FTIRRAS spectrum of MUAM in the mid-IR region is shown in FIG. 3,plot (A). The small peak centered at 1545 cm⁻¹ has been assigned as theNH₃ ⁺ deformation. The presence of this peak suggests that after arinsing with ethanol and Millipore water (pH˜6), a significant portionof the terminal amine groups exist in the protonated form. Variation inthe intensity of the 1545 cm⁻¹ peak can be effected by rinsing thesurface in solutions of differing pH. Bands at 1465 and 1258 cm⁻¹ inthis same plot have been assigned to the CH₂ scissoring and twistdeformations of the alkane chains respectively. The frequencies of thepeaks due to the CH₂ asymmetric stretching mode at 2923 cm⁻¹ and the CH₂symmetric stretching mode at 2853 cm⁻¹ (spectrum not shown) indicatethat the monolayer exists in a relatively ordered state. Absent from thespectrum in the CH stretching region is a band due to the N—H stretch(˜3200–3500 cm⁻¹) of the amine groups; it is assumed that this band istoo weak to be detected. Due to its terminal amine groups, a MUAMmonolayer surface is quite hydrophilic, which is verified by a contactangle measurement of 36.2±2.5 and is consistent with monolayerformation. Ex situ scanning SPR was used to measure a thickness of 17.5Å±0.4 Å for a gold surface modified with MUAM; this thickness isconsistent with that expected for a fully extended MUAM monolayeroriented nearly normal to the surface.

Step (2). In step (2) of the array fabrication, the MUAM covered surfaceis reacted with a reversible protecting group to create a hydrophobicsurface. In the case of MUAM, an amine-modified alkanethiol, theprotecting group is, appropriately, an amino protecting group,preferably Fmoc. Fmoc is a bulky, hydrophobic, base labile, amineprotecting group routinely used in the solid phase synthesis ofpeptides. The choice of protecting group used is dependent in largemeasure upon the nature of the ω-modification made to the alkanethiol.If the ω-modification is the addition of a carboxyl group, a hydrophobiccarboxy protecting group would be used. Likewise, if the ω-modificationis the addition of a hydroxyl or thiol group, a hydrophobic hydroxy orthiol protecting group, respectively, would be used. Any type ofhydrophobic protecting suitable for protecting the ω-modification usedon the alkanethiol can be utilized in the present invention. Numeroussuch protecting groups, for any number of reactive moieties, such asamine, hydroxy, and carboxy functionalities, are known to the art. Forexample, chloride derivatives of both Fmoc and trityl to can be used toreversibly modify hydroxyl-terminated alkanethiols.

The specific chemical reaction for Fmoc is shown in FIG. 2, referencenumber (2). The N-hydroxysuccinimide ester of Fmoc (Fmoc-NHS) reactswith the terminal amine moiety of the MUAM molecule to form a stablecarbamate (urethane) linkage, covalently attaching the Fmoc group to thesurface. The IR spectrum of Fmoc linked to a MUAM-coated gold substrateis shown in FIG. 3, plot (B). This spectrum provides evidence that thesurface reaction proceeds as expected. Prominent peaks at 1720, 1544,and 1267 cm⁻¹ are due to the carbamate (urethane) linkage which tethersthe Fmoc group to the MUAM surface. (The band at 1720 cm⁻¹ has beenassigned to the carbonyl stretching vibration (amide I), that at 1544cm⁻¹ to the CHN group vibration, and that at 1267 cm⁻¹ to the coupledC—N and C—O stretches (amide IV).) The peak at 1450 cm⁻¹ is ascribed tothe C═C ring stretch of the fluorenyl group and the band centered at1147 cm⁻¹ is attributed to the Fmoc C—O—C (ether) stretch. Afterreaction with Fmoc-NHS, the surface properties of the array are changedsignificantly; the surface is extremely hydrophobic as confirmed by themeasured contact angle of 74.4±2.5. In addition, an increase in the filmthickness to 22.8 Å±0.5 Å is measured with scanning angle SPR.

Step (3). In step (3) the bond anchoring the ω-modified alkanethiol tothe metal substrate is selectively cleaved to yield a patterned surfaceof exposed metal. UV photopatterning is preferred to create thepatterned surface, although the means to create the patterned surface isnot critical so long as the method reliable yields the desired pattern.For example, microcontact printing methods can also be used to yield apatterned surface. Using UV patterning, the surface is exposed through aquartz mask to UV radiation which photo-oxidizes the gold-sulfur bondthat anchors the alkanethiol monolayers to the surface. The surface isthen rinsed, removing the photo-oxidized alkanethiol and leaving anarray of bare metal pads surrounded by a hydrophobic MUAM+Fmocbackground. Using photopatterning, features with dimensions as small as50 mm have been achieved; using microcontact printing methods, arrayswith features as small as ˜100 nm are achievable.

Step (4). In step (4), the surface is again exposed to an ω-modifiedalkanethiol solution (in the preferred embodiment an ethanolic solutionof MUAM) whereby the alkanethiol assembles into the bare gold regionsproducing a surface composed of hydrophilic MUAM pads surrounded by thehydrophobic Fmoc background. This difference in hydrophobicity betweenthe reactive MUAM regions and the background is essential for thepinning of small volumes of aqueous biomolecule or cell solutions ontoindividual array locations.

Step (5). At step (5) in the process, biomolecules or cells (preferablynucleic acids) are then covalently attached to the surface. Asillustrated, the MUAM reactive pads are first exposed to a solution of abifunctional linker. To be used in the invention, the linker must becapable of binding at one end to the ω-modified alkanethiol surface andat the other end to the biomolecule or cell to be immobilized to formthe desired array. Any bifunctional linker having these characteristicscan be used in the present invention. The preferred bifunctional linkeris SSMCC, a heterobifunctional linker which contains both anN-hydroxysulfosuccinimide (NHSS) ester and a maleimide functionality.The NHSS ester end of the molecule reacts with the free amine groups onan amino-modified surface, such as the MUAM spots, creating padsterminated in maleimide groups which are reactive towards thiols. Smallvolumes (0.08 to 0.1 L) of 1 mM solutions of 5′-thiol-modified DNAsequences are then spotted at discrete array locations and react to forma covalent attachment to the surface. See FIG. 2, reference numeral (5).Using this technique, a whole host of biomolecules and/or whole cellscan be spotted at different array locations.

A variation on this attachment scheme whereby thiol-DNA is linked viaSSMCC to a MUA/PL (11-mercaptoundecanoic acid/poly-L-lysine) bilayer hasbeen used quite extensively in this laboratory, see U.S. Pat. No.5,629,213. Other researchers have used the direct self-assembly ofthiol-terminated DNA molecules on gold to prepare functionalizedsurfaces, but this method has the disadvantage that only weak forcesexist for the self-assembly of oligonucleotide molecules and hence, theDNA can also non-specifically adsorb to the bare gold surface.

Here, a bifunctional linker is used to attach 5′-thiol-modifiedoligonucleotide sequences to reactive pads of aminoalkanethiol. Thebifunctional linker preferably contains a functionality reactive towardsamines and a functionality reactive towards aminoalkanethiols. Thesurface is first exposed to a solution of the linker, whereby one end ofthe molecule reacts with the aminoalkanethiol surface. Excess linker isrinsed away and the array surface is then spotted with 5′-thiol-modifiednucleic acid which reacts with the other end of the bifunctional linker,forming a covalent bond between the nucleic acid and the surfacemonolayer.

Step (6). In step 6 the protecting group, depicted here as Fmoc isremoved from the array surface. Preferably, this is accomplished byexposure to a 1M solution of the secondary amine, TAEA, in DMF. Manybasic secondary amines can be used to remove Fmoc from the surface; forexample, 1M solutions of ethanolamine and piperidine can be used withequal success. TAEA was chosen specifically as the deprotection agentsince it effectively scavenges the dibenzofulvene byproduct and isefficiently rinsed from the array surface. After this deprotection step,the array background has been converted back to the original ω-modifiedalkanethiol surface. The spectrum of a deprotected MUAM surface, thepreferred embodiment, is shown in FIG. 3, plot (C); note the strongsimilarity between it and the original MUAM spectrum. The prominentbands due to the carbamate linkage no longer appear, indicating that theFmoc protecting group has been completely removed from the surface. Thedeprotected surface was also measured with scanning SPR, the thicknessmeasured was within ±1 Å of that measured for the starting MUAM surfaceand this gives additional proof that the Fmoc protecting group isremoved completely from the surface.

Step (7). In the final step of the array fabrication, the ω-modifiedalkanethiol background is reacted with a compound to create a backgroundthat is resistant to the non-specific binding of proteins. The preferredcompound for this purpose is PEG-NHS, although any compound which willselectively bind to the ω-modified alkanethiol surface and inhibitnon-selective protein binding can be used. In order to effectivelymonitor the binding of proteins to arrays of surface-bound biomoleculesor cells, it is critical that the array background prohibit thenon-specific adsorption of protein molecules. Significant amounts ofsuch non-specific binding obscures the measurement of small amounts ofprotein binding at specific array locations.

To create a background that is resistant to the non-specific binding ofproteins, the MUAM surface was reacted with PEG-NHS as is shown in FIG.2 reference number (7). As was the case in the Fmoc-NHS+MUAM reaction,PEG-NHS reacts with the terminal amine groups of the MUAM to form anamide linkage, covalently attaching the PEG polymer chain to thesurface. The preferred PEG-NHS polymer has an average molecular weightof 2000 and contains one NHS ester moiety per molecule, allowing for asingle point of attachment. The spectrum collected for a MUAM surfacereacted with PEG-NHS is shown in FIG. 3, plot (D). The peaks whichappear at 1660 cm⁻¹ and 1576 cm⁻¹ have been assigned as amide I and IIbands, respectively. The bands at 1457 cm⁻¹ and 1250–1260 cm⁻¹ areascribed to the scissoring and twist deformations of the CH₂ groupscontained in both the MUAM alkyl chains and the ethylene glycol (EG)groups. The band at 1352 cm⁻¹ is due to an EG CH₂ wagging mode, and theband centered at 1148 cm⁻¹ is due to the C—O—C (ether) stretch of theethylene glycol units. After the reaction of the deprotected surfacewith PEG-NHS, the surface remains hydrophilic and has a measured contactangle of 37.3±2.6. A total thickness of 23.8 Å±0.8 Å was measured for aMUAM monolayer film after reaction with PEG-NHS. This increase of only 6Å of PEG suggests that only a small fraction of the amine groups of theMUAM are modified and that the oligo(ethylene glycol) chains are lyingflat across the surface.

An SPR imaging experiment (see Example 2 and FIG. 7) was used to measurethe non-specific adsorption of BSA to a dual component surface(C₁₈-thiol/MUAM+PEG) and shows quite clearly that MUAM+PEG effectivelyresists the nonspecific adsorption of proteins.

EXAMPLES

The following Examples are included solely to provide a more completeunderstanding of the present invention. The Examples do not limit thescope of the invention disclosed and claimed herein in any fashion.

Standard Procedures for All Examples:

Gold substrates used in PM-FTIR and contact angle measurements werepurchased commercially (Evaporated Metal Films) and those used inscanning or imaging SPR measurements were prepared by vapor depositiononto microscope slide covers that had been silanized with(3-mercaptopropyl)trimethoxysilane (Aldrich) in a manner similar to thatreported by Goss et al. (1991) Anal. Chem. 63:85–88.

All oligonucleotides were synthesized on an ABI (Foster, Calif.) DNAsynthesizer at the University of Wisconsin Biotechnology Center. GlenResearch's (Sterling, Va.) “5′-Thiol-Modifier C6” and ABI's “6-FAM” wereused for 5′-thiol-modified and 5′-fluorescein-modified oligonucleotidesrespectively, and “Spacer Phosphoramidite 18” (Glen Research) was usedfor the addition of an ethylene glycol spacer region. Thiol-modifiedoligonucleotides were deprotected as outlined by Glen Research's productliterature.(Glen Research Corp. (1990) “User Guide to DNA Modificationand Labeling”). Before use, each oligonucleotide was purified byreverse-phase binary gradient elution HPLC (Shimadzu (Columbia, Md.)“SCL-10AVP”) and DNA concentrations were verified with an HP8452A UV-VISspectrophotometer (Hewlett-Packard, Palo Alto, Calif.).

The sequences of the DNA molecules used in the SSB experiment of Example1 were as follows:

D1=5′ HS(CH₂)₆(T)₁₆AAC GAT GCA GGA GCA A (SEQ. ID. NO: 1)

D2=5′ HS(CH₂)₆(CH₂CH₂O)₂₄GCT TAT CGA GCT TTC G (SEQ. ID. NO: 2)

-   -   D2 complement=5′FAM-CGA AAG CTC GAT AAG C (SEQ. ID. NO: 3)

The buffer used in the BSA and SSB SPR imaging experiments contained 20mM phosphate, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5 mM MgCl₂ and wasbuffered at pH 7.4.

Multi-step array fabrication: A clean gold substrate was immersed in a 1mM ethanolic solution of MUAM for at least one hour to allow for theadsorption and self-assembly of the aminoalkanethiol monolayer. Thesubstrate was rinsed with ethanol and water, dried under a stream of N₂,and was then reacted with a solution of Fmoc-NHS (3 mM in 1:1 DMSO:100mM TEA buffer, pH 7). The sample was soaked briefly in DMSO to removeunreacted Fmoc-NHS from the surface and then photopatterned byirradiating the sample with UV light from a mercury-xenon arc lampthrough a quartz mask. Subsequent rinsing of the sample with ethanol andwater removed alkanethiol from the exposed areas. The sample wasre-exposed to the ethanolic MUAM solution resulting in an array of MUAMelements surrounded by a hydrophobic MUAM+Fmoc background.Single-stranded, 5′-thiol modified DNA was then immobilized onto thearray locations using an attachment scheme modified slightly from thatused previously. Briefly, the amine-terminated MUAM array elements werespotted with 0.1 μL of a 1 mM solution (in 100 mM TEA, pH 7) of theheterobifunctional linker SSMCC, creating a thiol-reactive,maleimide-terminated surface. 5′-Thiol-modified DNA sequences were thencovalently attached to these maleimide-terminated array elements byspotting the sample with 0.1 μL drops of solutions containing 1 mM DNAonto the specific array locations and reacting for at least 2 hours in ahumid environment to prevent solvent evaporation. After exposure to theDNA solution, the surface was rinsed with water and soaked in buffer toremove unbound DNA sequences. The Fmoc was then removed from thebackground by immersing the array in a 1M solution of TAEA in DMF for 10minutes. The deprotected surface was rinsed with water and subsequentlyreacted with 4 mM PEG-NHS (in 100 mM TEA, pH 8) to pegylate the arraybackground, rendering it resistant to protein non-specific binding.

PM-FT-IRRAS Measurements: PM-FT-IRRAS spectra were collected on aMattson RS-1 spectrometer equipped with either a narrow band HgCdTedetector (for spectra in the mid-IR region, 2000–1000 cm⁻¹) or an InSbdetector (for spectra in the CH stretching region, 3400–2600 cm⁻¹). Theoptical layout and previously developed real-time interferogram samplingmethods have been described elsewhere and need not be elaborated uponhere. The PM-FT-IRRAS differential reflectance values (% RIR) wereconverted to absorbance units for comparison with conventional IRRASdata. Spectra are an average of 1000 scans collected at 2 cm⁻¹resolution.

Contact Angle Measurements: Water contact angles were determined atambient laboratory temperatures using standard and well knownprocedures. Ten microliter droplets were dispensed from a Gilson pipetteonto the surface and the angle measurement was recorded immediately.Reported contact angle values for both the Fmoc and PEG functionalizedsurfaces are the average of 12 different measurements taken on 4individually prepared samples and the value for MUAM is the average of30 measurements taken on 10 different samples.

Scanning Angle SPR Measurements: The-optical technique of ex situscanning SPR was used to determine the thickness (reported hereinabove)of MUAM, MUAM+Fmoc, and MUAM+PEG assembled on BK7 coverslips (FisherScientific, Pittsburgh, Pa.) onto which 475 Å of Au was vapor deposited.Details of the SPR experiment and thickness calculations have beenreported elsewhere. Briefly, the reflectivity (R) of a p-polarized HeNelaser beam (632.8 nm) from a sample assembly (BK7 prism/Au/thinfilm/air) is monitored as a function of incident angle, to generate aSPR curve (% vs. angle). A steep drop in the reflectivity occurs atangles just past the critical angle (˜44). The exact position of theminimum is determined by the thickness and index of refraction ofmaterial adsorbed at the gold surface. A 4-phase complex Fresnelcalculation was used to determine the film thickness and a refractiveindex of 1.45 was assumed for all the thin films measured here.

SPR Imaging Apparatus: The in situ SPR imaging instrument is a modifiedversion of that described previously, see Jordan & Corn (1997) Anal.Chem. 69(7):1449–1456; Thiel et al. (1997) Anal. Chem. 69:4948–4956;Jordan et al. (1997) Anal. Chem. 69(24):4939–4947; and Frutos et al.(1998), supra, in which the HeNe laser and beam expander have beenreplaced by a collimated white light source/bandpass filter combination.A more thorough discussion of this modification in the context of nearIR (NIR) SPR imaging is reported elsewhere, see Nelson et al. (1999), inpreparation. In short, a collimated, polychromatic beam of light wasused to illuminate an SF10 prism/Au/thin film/buffer assembly at a fixedincident angle near the SPR angle. The reflected light was passedthrough a 10 nm bandpass filter (830 nm) and was collected with aninexpensive CCD camera. Differences in the reflected light intensitymeasured at various locations on the sample create the image and are adirect result of differences in the thickness or refractive index of thematerial bound at the gold surface. The image shown in FIG. 6 wascollected in situ for a sample constructed on SF10 substrates onto which450 Å of Au had been deposited. Data work-up was done using NIH Imagev.1.61 software.

Example 1

SPR Imaging Measurements of the Binding of Single Stranded DNA BindingProtein to Arrays of Single and Double-stranded DNA Sequences:

To demonstrate that nucleic arrays can be used in conjunction withimaging SPR to monitor protein-nucleic acid binding, a checkerboardsurface was constructed containing both single-stranded DNA (D1, SEQ.ID. NO: 1) and double-stranded DNA (D2 and its complement, SEQ. ID. NOS.2 and 3, respectively), by the methods described immediately above. Thebinding of single-stranded DNA binding protein, SSB, to the arraysurface was then monitored by SPR. As its name implies, SSB (a tetramerof four identical subunits with a total molecular weight of 75,000 D)binds tightly, selectively, and cooperatively to single-stranded DNA andplays a central role in DNA replication, repair, and recombination. FIG.6 shows the difference between two images collected immediately beforeand after the exposure of the surface to SSB. The raised areas on theimage are a measure of the change in % R upon adsorption of the proteinto the surface. The array locations at which the protein boundcorrespond to those regions which were modified with single-stranded DNAsequences.

FIG. 5 shows various line profiles taken from images collected duringthe course of the experiment. These “line profiles,” which providequantitative information, are constructed by averaging the % R valuesmeasured for each column of pixels in a selected rectangular regiondrawn across the image and then plotting this average value against thatcolumn's lateral position. The solid line shows the starting surface inwhich two 5′-thiol-modified, single-stranded DNA sequences, D1 and D2,were immobilized in a checkerboard pattern onto the array surface. Thesequences of these two DNA probe strands are noted above. Each sequencecontains a 5′-thiol modifier, a spacer region, and a 16 base-longvariable sequence. The variable regions were specifically chosen from alibrary developed for the purposes of DNA computing, Frutos et al.(1997) supra, they and their complements exhibit no cross hybridization.To position the DNA sufficiently far from the surface so that stearichindrance does not interfere with the hybridization adsorption process,a spacer region is incorporated. A 15T spacer region was used for D1,but sequence D2 contained a similar length EG spacer instead. This wasnecessary given the fact that SSB is known to bind quite strongly topolyT sequences. The dashed line shows the effects of exposing thesurface, in situ, to a solution containing the 16-mer complement to D2(SEQ. ID. NO: 3). A measurable change in % R occurred at location D2,indicating that hybridization adsorption of the complementary sequencetook place; no increase in signal was seen at the D1 locations. Thedot-dashed line shows the surface after exposure to a 200 nM solution ofSSB. As expected, the protein bound strongly to locations on the arraywhich were single stranded but also bound slightly to those locationsthat contained double-stranded sequences. Since SSB does not bind todouble-stranded DNA, we attribute the increased signal at location D2 tothe binding of SSB to single-stranded DNA present at these locations asa result of incomplete hybridization. It is important to note that thearray background successfully resisted the non-specific binding of bothcomplementary DNA molecules and single-stranded binding protein; thisallows the measurement of small changes in % R without interference froma high background signal.

This Example shows that the arrays fabricated according to the presentinvention can be used to probe nucleic acid-protein interactions.

Example 2

Demonstration that PEG Block Non-specific Protein Binding:

Here, an array of C₁₈ alkanethiol spots were assembled on a MUAM+PEGbackground using the techniques described above. The array was thenexposed to BSA and the percent reflectivity measured as described inExample 1. The results are shown in FIG. 7. In FIG. 7, the solid lineshows the percent reflectivity of the array prior to exposure to BSA.The dashed line shows the presence reflectivity after exposure to BSA.

While some BSA did adsorb to the MUAM+PEG background, a vastly largeamount of BSA adsorbed to the untreated C₁₈ spots. This Example thusshows that PEG can be used to inhibit the non-specific binding ofproteins to the background surface.

1. A biomolecule or cellular array on a metal substrate, the arraycomprising: a metal substrate; a continuous layer of an identicalω-modified alkanethiol adhered to the metal substrate; one or morediscrete spots of biomolecules or cells directly bonded to thecontinuous layer of ω-modified alkanethiol; and wherein areas of thecontinuous layer of ω-modified alkanethiol not covered by one of thediscrete spots are covered by a background material resistant tonon-specific protein binding.
 2. The array of claim 1, wherein the spotsare DNA spots.
 3. The array of claim 1, wherein the spots are RNA spots.4. The array of claim 1, wherein the background material comprisespoly(ethylene glycol) moieties.
 5. The array of claim 1, furthercomprising a bifunctional linker interposed between each biomolecule orcellular spot and the ω-modified, alkanethiol-coated metal substrate. 6.The array of claim 1, wherein the metal substrate is gold.
 7. The arrayof claim 1, wherein the substrate is selected from the group consistingof noble metals.
 8. The array of claim 1, wherein the substrate isselected from the group consisting of gold, silver, platinum, copper,cadmium, and aluminum.
 9. The array of claim 1, wherein the substrate issilver.
 10. The array of claim 1, wherein the spots are cells.
 11. Thearray of 5, wherein the bifunctional linker is a heterobifunctionallinker.
 12. The array of claim 11, wherein the heterobifunctional linkeris sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.